DESCRIPTION (adapted from candidate's abstract): Synaptic transmission is initiated by the release of neurotransmitters into the synaptic cleft. Release is regulated by several mechanisms that probably involve phosphorylation. The candidate proposes to study the role of synaptic protein phosphorylation in the regulation of the release of GLU, an excitatory neurotransmitter. To carry out studies where both the molecular components and physiological consequences of a phosphorylation event can be evaluated, the candidate will use protein chemistry, recombinant DNA techniques and functional assays. Specific Aim 1 of the proposal is to establish a preparation which is suitable for pharmacological manipulations of protein kinase (PK) activities, release studies and biochemical measurements. The candidate proposes to use hippocampal CA3 synaptosomes that are enriched in mossy fiber (mf) nerve endings and CAl nerve terminals, respectively. Preliminary studies suggest that calcium-calmodulin-dependent-protein kinases (CaM K) enhance release from both preparations, while PKA potentiates release only from mf enriched synaptosomes. Specific Aim 2 is to identity synaptic phosphoproteins which are targets for PKA and CaM KII under the experimental conditions which modulate GLU release. Immuno-precipitation of 32P-labeled synaptosomes will be used to detect phosphorylation of synaptic proteins. Preliminary data indicate that rabphilin-3a and RIM, proteins of the release machinery, undergo mf specific PKA-dependent increase in phosphorylation. These findings established rabphilin-3a and RIM as candidate phosphoproteins whose phosphorylation plays an important role in PKA-dependent up-regulation of GLU release. Specific Aim 3 is to test the physiological relevance of the phosphorylation of rabphilin-3a/RIM and other proteins that will have been identified in the above experiments, by loading cultured neurones mutated at the phosphorylation site, co-transfecting PC-12 cells with these mutant proteins, or using synaptosomes from mice lacking the protein of interest. Completion of this proposal will enhance our understanding about the regulatory mechanisms of neurotransmitter release and will likely lead to advances in the diagnosis and treatment in neurological diseases. In addition, this project will provide training in molecular neuroscience for the candidate and broaden his expertise.